piRNA loading triggers MIWI translocation from the intermitochondrial cement to chromatoid body during mouse spermatogenesis

The intermitochondrial cement (IMC) and chromatoid body (CB) are posited as central sites for piRNA activity in mice, with MIWI initially assembling in the IMC for piRNA processing before translocating to the CB for functional deployment. The regulatory mechanism underpinning MIWI translocation, however, has remained elusive. We unveil that piRNA loading is the trigger for MIWI translocation from the IMC to CB. Mechanistically, piRNA loading facilitates MIWI release from the IMC by weakening its ties with the mitochondria-anchored TDRKH. This, in turn, enables arginine methylation of MIWI, augmenting its binding affinity for TDRD6 and ensuring its integration within the CB. Notably, loss of piRNA-loading ability causes MIWI entrapment in the IMC and its destabilization in male germ cells, leading to defective spermatogenesis and male infertility in mice. Collectively, our findings establish the critical role of piRNA loading in MIWI translocation during spermatogenesis, offering new insights into piRNA biology in mammals.

The PDF file includes         (lanes 7-12) with thermolysin.The reactions were carried out in the absence of thermolysin (lanes 1 and 7) or in the presence of increasing amounts of thermolysin (lanes 2-6 and 8-12) before immunoblotted using anti-MIWI antibody.d Ubiquitination of MIWI detected in adult wildtype, Miwi YY/YY and Miwi YK/YK mice.Top, anti-MIWI IP pellets (lanes 2-4) were immunoblotted by anti-Ub for Ub-MIWI conjugates, using IgG IP (lane 1) as a negative control.Bottom, anti-MIWI IB provided a loading reference.Results shown are representative of three independent experiments.Source data are provided as a Source Data file.

Fig
Fig. S5 | TDRD6 depletion does not affect MIWI and piRNA expression in mice.

Fig. S6 |
Fig. S6 | TDRD6 depletion impairs MIWI integration into the CB during male germ cell differentiation in mice.

Fig. S1 |
Fig. S1 | The construction of piRNA loading-deficient Miwi YY/YY and Miwi YK/YK mice.a schematic diagram illustrating the construction of Miwi YY/YY (left) and Miwi YK/YK mutant mice (right) using CRISPR/Cas9.Top, genomic structure of Miwi and sequences of guide RNA and donors; bottom, Sanger sequencing of genotyping PCR products amplified from the guide RNA-targeted area to confirm the mutant alleles.b Detection of potential off-target effects of the guide RNAs in Miwi YY/YY and Miwi YK/YK mutant mouse genome by PCR amplification and Sanger sequencing.

Fig. S3 |
Fig. S3 | Immunostaining of MILI and MVH in testis sections from Miwi mutant mice.a, b Immunostaining of MILI (a, red) and MVH (b, red) on testis sections from adult wildtype, Miwi YY/YY , Miwi YK/YK and Miwi -/-mice using regular microscopy.Left: representative staining images of indicated mouse testis sections, scale bar, 20 µm; right, the enlargement of yellow framed regions, scale bar, 5 µm.The developmental stages of spermatocytes and spermatids were distinguished according to γH2AX (green) and DAPI (greyscale) staining.RS, round spermatids.Results shown are representative of three independent experiments.

Fig. S4 |
Fig. S4 | Validation of the specificity anti-MIWI unloaded antibody.a Western blotting of MIWI protein using anti-MIWI unloaded antibody in Flag-tagged wildtype MIWI or epitope-deleted MIWI Δaa701- 801 mutant-transfected HEK293T cells (lanes 1 and 2), and adult wildtype or Miwi -/-testes (lanes 4 and 5), with β-actin serving as a loading control.b Anti-MIWI unloaded preferably pulled down piRNAunloaded MIWI in Flag-MIWI-transfected HEK293T cells.RNA co-IP assays using anti-MIWI unloaded and control anti-MIWI antibodies in cell lysate incubated with Cy5-labeled piRNA, with anti-MIWI IB as loading references.c Western blotting of MIWI protein in parental and Flag-tagged MIWI-stableexpressed GC-2spd (ts) cells, with β-actin serving as a loading control.Results shown are representative of three independent experiments.Source data are provided as a Source Data file.

Table S1
Oligonucleotides and antibodies used in this study.